Progesterone receptor (PR) dependent regulation of target genes requires protein/protein interactions for the recruitment of coactivators and in some cases for interaction of PR with target genes. Understanding the regulation of these complex interactions is the next important step in elucidating PR function;phosphorylation is a well established means of regulating protein/protein interactions. The goal of this collaboration between the Weigel and Edwards labs is to understand how cell signaling and protein phosphorylation regulates the activity of PR. Our central hypothesis is that site specific PR phosphorylation regulates protein/protein interactions that are required for regulation of subsets of PR target genes and that phosphorylation is integral to accomplishing the biological actions of PR. To test this, we will: 1: test the hypothesis that there is a "phosphorylation code" for PR resulting in differential regulation of target genes depending upon the phosphorylation status of PR. 2: Determine the means by which selected phosphorylations modulate PR function. Specific Aim 3: Test the concept that selective elimination of a PR phosphorylation site will compromise PR function in mice. Specific Aim 4: determine the role of PR phosphorylation in mammary gland differentiation and function in vivo by use of a "tissue reconstitution" system. We will use microarray analyses to identify genes differentially regulated by PR phosphorylation site mutants, chromatin immunoprecipitation (ChIP) on chip to identify PR binding sites in genes of interest followed by biochemical analyses for the mechanisms for differential regulation (binding of PR and interaction with transcription factors or differential recruitment of cofactors). To test for the biological relevance of phosphorylation, we will (1) engineer a mouse expressing PR lacking one phosphorylation site and test the biological consequences of this substitution (2) use a novel mouse mammary gland reconstitution approach to test the biological contributions of additional phosphorylation sites. These studies will be the first to comprehensively assess the role of individual phosphorylations in steroid receptor mediated gene regulation and the biological contributions of phosphorylation to receptor function. Significance: PR action is beneficial in uterus, but may be a risk for breast cancer. Our studies will provide a better understanding of how PR function is regulated: this information can be used to develop means to selectively regulate PR function for medical applications.